Significance: The ultimate goal of this project is a CE-marked, FDA-cleared and CLIA-waived rapid (<20 min) smartphone-based point-of-care (POC) test to aid the diagnosis of Chlamydia trachomatis (CT) infections. CT infections are the most common bacterial STI, with almost 3 million new cases in the US each year, and associated costs exceeding $2.4 billion. Untreated women, mostly in the 15-24 age group, are at a high risk of serious sequelae including pelvic inflammatory disease, ectopic pregnancy, and infertility. CT infections are curable with a single pill, but the fact that they are asymptomatic in up to 9 out o 10 infected men and women necessitates screening of high-risk populations using accurate testing methods. Currently used Nucleic Acid Amplification Techniques (NAATs) are expensive and have a significant delay between specimen collection and result, severely limiting their applicability in disproportionately affected resource poor settings. Rapid POC tests can prevent sequelae and curb disease transmission by enabling a same-day test and treat approach and immediate partner notification. Current POC tests for CT, based on the lateral flow test (LFT) and using colloidal gold as reporters, are > 95% specific, but have low clinical sensitivities (down to 27%) due to their poor (high) limit of detection (LOD). In this project, Luminostics' proprietary nanophosphors, silica- encapsulated persistent luminescence nanoparticles, will be used as LFT reporters in an immunoassay for Chlamydial LPS (cLPS). Upon assay completion (<15 min), the disposable LFT cartridge will be readout by a smartphone using an inexpensive phone case-like attachment and software app, making it extremely field- deployable and attractive for home testing. Hypothesis: We hypothesize that the improved LOD of nanophosphor-LFTs, expected to be 20- to 200-fold lower than colloidal gold-LFTs, will result in clinical sensitivities approaching NAATs, which are >95% sensitive. Preliminary results: Using the same affinity reagents, a 20-fold improvement in analytical sensitivity of nanophosphor-LFTs over colloidal gold-LFTs has been demonstrated using unoptimized and polydisperse nanophosphors, as has the feasibility of highly sensitive luminescence detection using an iPhone. Specific Aims: The Phase I deliverable of this project will be an analytically and clinicaly validated device prototype. In Aim 1, nanophosphor-LFT parameters and smartphone components will be optimized for sensitive CT detection. In Aim 2, the performance of the optimized device prototype will be evaluated through analytical sensitivity, specificity, and interference studies, and through a small-scale retrospective clinical study. The success of this Phase I project, the odds of which are maximized by collaborations with or the technical services of surface chemistry, STI, LFT, and cLPS experts, will set the stage for device integration and prospective clinical testing of the new POC test for CT in Phase II. It will also validate nanophosphor-LFTs as a platform technology for ultrasensitive analyte detection, with potential applications in a variety of critical medical conditions that need more sensitive POC tests.